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Chicken embryo extracts contain a factor that preferentially blocks the accumulation of RNA polymerase II transcripts
Abstract:
Chick embryos, chick embryo fibroblasts, and Rous sarcoma virus-transformed chick embryo fibroblasts contain a factor that preferentially blocks the accumulation of DNA-directed RNA polymerase II transcripts. The factor was detected by inhibition of transcription in a cell-free assay system utilizing partially purified RNA polymerase II from calf thymus, soluble factors from HeLa cells, and a purified DNA template. At low concentrations, it specifically prevents the accumulation of RNA polymerase II transcripts; at higher concentrations, it blocks the accumulation of other transcripts. The factor has been partially purified by sequential chromatography on BioRex 70, DNA-cellulose, Bio-Gel P-6, and HPX-87 from extracts of chicken embryos. The activity was resistant to treatment with trypsin, pronase, or micrococcal nuclease. A partial characterization of the molecule indicates that (i) it has an apparent molecular mass of about 200-300 daltons, (ii) it is stable at pH 2 and pH 12 and to heating at 100 degrees C, (iii) it is not extractable by ether or chloroform:methanol, (2:1, v/v), and (iv) it is labile to heating at 800 degrees C. These data suggest that it is a small, hydrolphilic compound probably organic in nature. The factor is active in a transcription assay utilizing either the Rous sarcoma virus Long Terminal Repeat promoter or the chick alpha 2 (Type I) collagen-promoter as DNA templates. The accumulation of promoter-specific transcripts is blocked in a cell-free assay utilizing either Rous sarcoma virus-chick embryo fibroblast extracts or HeLa S-100 factors and calf thymus RNA polymerase II. In the absence of S-100, the factor does not appreciably affect the accumulation of randomly initiated transcripts produced by calf thymus RNA polymerase II on a DNA template; this result indicates the factor interacts directly or indirectly with some component(s) of HeLa S-100 to prevent the accumulation of RNA.
Insights
A novel factor in chick embryos blocks RNA polymerase II transcription. This small, stable molecule specifically inhibits promoter-driven RNA accumulation, suggesting a role in gene regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Chick embryos and fibroblasts contain a transcription-inhibiting factor.
- This factor preferentially affects DNA-directed RNA polymerase II transcripts.
Purpose of the Study:
- To detect, purify, and characterize a novel transcription inhibitory factor from chick embryos.
- To elucidate the factor's specificity and molecular properties.
Main Methods:
- Cell-free transcription assays using partially purified RNA polymerase II and DNA templates.
- Sequential chromatography for factor purification.
- Biochemical characterization including protease and nuclease treatments, molecular weight estimation, and stability tests.
Main Results:
- A factor was identified that inhibits RNA polymerase II transcript accumulation.
- The factor is small (200-300 daltons), stable to heat and pH extremes, and resistant to proteases/nucleases.
- It specifically blocks promoter-driven transcription, interacting with HeLa S-100 cell components.
Conclusions:
- A novel, small, hydrophilic organic compound inhibits specific RNA transcription in vitro.
- The factor's properties suggest a regulatory role in gene expression.
- Further research is needed to identify the precise molecular target and in vivo function.